Fascins is a widely distributed protein family. It can concentrate F-actin filaments into bundles. (Edwards, R. A. et al., 1995 Cell Motil. Cytoskeleton 32, 1-9). The genes of fascins have been cloned from human, mice and Xenopus. (Duh, F. M. et al., 1994, DNA Cell Biol. 13:821-827; Edwards, R. A. et al., 1995, J. Biol. Chem. 270, 10764-10770; Holthuis, J. C. M. et al., 1994 Biochim. Biophys. Acta 1219: 184-188). As actin-binding protein, each fascin is expressed in many cellular structures such as micropodia and filopodia. The ubiquitous localization of fascins indicates their important function in morphogenesis of subcellular structures. In photoreceptor cells, actin is located in inner segment, synaptic terminal of the rod cells, the connecting cilium between inner segment and outer segment, the labial part of outer segment of rod cells, and the basal part of the rod outer segments. Actin plays an important role in these parts as to regulate morphogenesis of outer segment disk and in lower vertebrates shorten the photoreceptor to adjust to brightness (Williams, D. S. et al., 1988, J. Comp. Neurol. 272:161-176). As cell specific actin-binding proteins, fascins can help actin fulfill these functions.
There are three conservative domains in the fascin family of proteins. The largest one consist of twenty eight amino acid residues located in the N-terminal, and fifteen amino acid residues among these are almost completely conserved. The second one is the amino acid sequence ETFQLE in the middle of the full sequence. The third is the sequence GKYW near the C-terminal of the proteins.
Sequence analysis of a newly discovered protein reveals that it has all three conservative domains of the fascin family, with 92% of the conservative amino acids, and it is 95% homologous to human fascin. So the protein is a new member of fascin family. And because it is specifically expressed in photoreceptor cells, it was named retinal fascin (Yoshitsugu, S. et al., 1997 FEBS Lett. 414(2):381-6).
In addition to the three conservative domains, retinal fascin also has three highly conservative amino acid of fascin family—Gly 409, Ser 289 and Ser 39. Change of the first two amino acid will lead to functional loss of retinal fascin (Cant, K. et al., 1996, Genetics 143, 249-258). Ser39 is probably a phosphorylation site and phosphorylation of retinal fascin is very important for the organization of microfilaments and cell mobility (Yamakita, Y. et al., 1996, J. Biol. Chem. 271:12632-12638). The full length cDNA of retinal fascin is 1589 bp, including an open reading frame encoding a 492 aa. protein with an apparent molecular weight 55070 Da. There are neither N-terminal signal sequence nor transmembrane domain in retinal fascin.
Besides Ser39, retinal fascin also has phosphorylation sites for protein kinase A, protein kinase C and tyrosine kinase. These sites are: nine phosphorylation sites for protein kinase C—Thr20, Ser39, Ser147, Thr237, Ser272, The300, Thr375, Thr404, Thr465; and two phosphorylation sites for protein kinase A—Thr304, Ser399; two phosphorylation sites for Tyrosine kinase—Tyr193, Tyr228. These indicate that retinal fascin is regulated by phosphorylation.
The polypeptide of the present invention is 29% identical and 44% homogenous to retinal fascin at the protein level and has the three conservative domains of fascin family. So the polypeptide of the present is named “retinal fascin54” and is found to have similar biological functions with retinal fascin.
As discussed above, human actin-binding protein p54 plays an essential role in the regulation of important biological functions such as cell division and embryogenesis, and it's believed that many of proteins are involved in these regulations. So the determination of those related human actin-binding proteins, especially of their amino acid sequences is always desired in this filed. The isolation of this novel human actin-binding protein p54 builds the basis for research of the protein function under normal and clinical conditions, disease diagnosis and drug development. So the isolation of its cDNA is very important.